Journal: The Journal of Physiology

Article Title: The vital role of arcuate nociceptin/orphanin FQ neurones in mounting an oestradiol‐dependent adaptive response to negative energy balance via inhibition of nearby proopiomelanocortin neurones

doi: 10.1113/JP283378

Figure Lengend Snippet: A–C , representative photomicrograph of mCherry fluorescing N/OFQ ARC neurones in low power (×4) ( A ), a high power image (×40) DIC image of a recorded N/OFQ neurone ( B ), and a high power (×40) image depicting its corresponding mCherry fluorescence ( C ). D–F , representative current clamp traces of N/OFQ ARC neurones obtained from transgenic PNOC‐ cre mice injected bilaterally with mCherry‐tagged, Gq‐coupled excitatory DREADD ( D ), mCherry control ( E ), and mCherry‐ tagged Gi‐coupled inhibitory DREADD ( F ). Current clamp traces depict the depolarization and hyperexcitability of firing associated with stimulation of N/OFQ ARC neurones, no change in membrane potential or firing without the association of DREADDs, and the hyperpolarization and cessation of firing associated with inhibition of N/OFQ ARC neurones. G , composite data depicting the significantly depolarizing change in membrane potential upon chemostimulation of N/OFQ ARC neurones, as well as the hyperpolarization seen upon chemoinhibition of these cells. Bars represent means, and lines 1 SD of the induced change upon application of CNO ( D ); one‐way ANOVA, # P < 0.05, relative to Hm4D(Gi), * P < 0.05 relative to mCherry blank control. Control n = 3 cells in 2 slices from 1 animal; excitatory DREADD n = 5 cells in 3 slices from 1 animal; inhibitory DREADD n = 3 cells in 2 slices from 1 animal. [Colour figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Cre recombinase‐dependent AAV vectors containing either cation channel rhodopsin‐2 (ChR2; Mattis et al., ; AAV1.EF1a.DIO.ChR2 [E123A].YFP.WPRE.jGH; 7.2 × 10 12 genomic copies/ml; 300 nl total volume; Addgene (Watertown, MA, USA) plasmid no. 35507), excitatory designer receptor exclusively activated by designer drug (DREADD; pAAV‐hSyn‐DIO‐hM3D(Gq)‐mCherry; 7×10 12 genomic copies/ml; 300 nl total volume; Addgene plasmid gifted by Bryan Roth, no. 44361), inhibitory DREADD (pAAV‐hSyn‐DIO‐hM4D(Gi)‐mCherry; 7 × 10 12 genomic copies/ml; 300 nl total volume; Addgene plasmid gifted by Bryan Roth, no. 44362), or their respective enhanced yellow fluorescent protein (eYFP; Mattis et al., ; pAAV‐Ef1a‐DIO EYFP; 1.0×10 13 ; 300 nl total volume; Addgene plasmid deposited by Karl Deisseroth, no. 27056) and mCherry blank controls (pAAV‐hSyn‐DIO‐mCherry; 7 × 10 12 genomic copies/ml; 300 nl total volume; Addgene plasmid gifted by Bryan Roth, no. 50459) were administered over a span of 2 min to double transgenic PNOC‐ cre /eGFP POMC mice, PNOC‐ cre mice and WT controls.

Techniques: Fluorescence, Transgenic Assay, Injection, Control, Membrane, Inhibition

Journal: The Journal of Physiology

Article Title: The vital role of arcuate nociceptin/orphanin FQ neurones in mounting an oestradiol‐dependent adaptive response to negative energy balance via inhibition of nearby proopiomelanocortin neurones

doi: 10.1113/JP283378

Figure Lengend Snippet: A–E , representative photomicrograph. A , low power (×4) image of eGFP‐fluorescing POMC ARC neurones taken from double transgenic PNOC‐ cre /eGFP‐POMC mice. B , low power (×4) image depicting either Gq mCherry tagged excitatory DREADD‐ or mCherry blank control‐expressing N/OFQ ARC neurones. C and D , differential interference contrast image (×40) of a recorded POMC neurone ( C ) and corresponding eGFP fluorescence from the same neurone ( D ). E , mCherry‐expressing N/OFQ ARC neurones within the vicinity of the recorded POMC ARC neurone. F–H , representative voltage clamp traces depicting the modest outward current in the ad libitum group elicited in POMC ARC neurones via activation of N/OFQ ARC neurones, and the robust outward current in the fasting group receiving the same treatment. Additionally, the mCherry blank control group displays no change in current, assuring that the outward currents are only elicited in cells from the DREADD‐injected animals, and therefore a consequence of stimulation of N/OFQ ARC neurones. I and J , current–voltage relationships between baseline and application of CNO, displaying the reversal potential for potassium in both groups and a greater change in slope conductance for the fasting group. K and L , composite data depicting the significantly greater change in current and slope conductance (measured between −60 and −80 mV) for POMC ARC neurones under conditions of negative energy balance as compared to the ad libitum group and mCherry blank control. Bars represent means and lines 1 SD. * P < 0.05 relative to control, # P < 0.05, one‐way ANOVA/LSD, relative to ad libitum . Control n = 13 cells in 7 slices from 2 animals; ad libitum n = 26 cells in 16 slices from 5 animals; fasted n = 24 cells in 14 slices from 5 animals. [Colour figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Cre recombinase‐dependent AAV vectors containing either cation channel rhodopsin‐2 (ChR2; Mattis et al., ; AAV1.EF1a.DIO.ChR2 [E123A].YFP.WPRE.jGH; 7.2 × 10 12 genomic copies/ml; 300 nl total volume; Addgene (Watertown, MA, USA) plasmid no. 35507), excitatory designer receptor exclusively activated by designer drug (DREADD; pAAV‐hSyn‐DIO‐hM3D(Gq)‐mCherry; 7×10 12 genomic copies/ml; 300 nl total volume; Addgene plasmid gifted by Bryan Roth, no. 44361), inhibitory DREADD (pAAV‐hSyn‐DIO‐hM4D(Gi)‐mCherry; 7 × 10 12 genomic copies/ml; 300 nl total volume; Addgene plasmid gifted by Bryan Roth, no. 44362), or their respective enhanced yellow fluorescent protein (eYFP; Mattis et al., ; pAAV‐Ef1a‐DIO EYFP; 1.0×10 13 ; 300 nl total volume; Addgene plasmid deposited by Karl Deisseroth, no. 27056) and mCherry blank controls (pAAV‐hSyn‐DIO‐mCherry; 7 × 10 12 genomic copies/ml; 300 nl total volume; Addgene plasmid gifted by Bryan Roth, no. 50459) were administered over a span of 2 min to double transgenic PNOC‐ cre /eGFP POMC mice, PNOC‐ cre mice and WT controls.

Techniques: Transgenic Assay, Control, Expressing, Fluorescence, Activation Assay, Injection

Journal: The Journal of Physiology

Article Title: The vital role of arcuate nociceptin/orphanin FQ neurones in mounting an oestradiol‐dependent adaptive response to negative energy balance via inhibition of nearby proopiomelanocortin neurones

doi: 10.1113/JP283378

Figure Lengend Snippet: A–C , representative photomicrographs. A , low power (×4) image of eGFP‐fluorescing POMC ARC neurones. B and C , high power (×40) DIC image of a recorded POMC ARC neurone ( B ) and the eGFP fluorescence of the same neurone ( C ). D and E , representative voltage clamp traces depicting the inward currents elicited by antagonism of the NOP receptor under sated, ad libitum ‐fed conditions ( D ), and the potentiation under negative energy balance ( E ). F , composite data depicting negative energy balance elicits a significantly greater change in current compared to ad libitum conditions. * P < 0.05 relative to ad libitum ; ad libitum n = 23 cells in 13 slices from 5 animals; fasted n = 18 cells in 12 slices from 5 animals; Student's t test. G–I , representative traces depicting chemogenetic inhibition of N/OFQ ARC neurones inducing a reversible inward current in ad libitum ( H ) conditions, which is potentiated under fasted conditions ( I ); inhibition of this system is dependent on presence of DREADD in N/OFQ ARC neurones, as there is no CNO‐induced change in current in the mCherry blank control group ( G ). J , composite data depicting inward currents are significantly greater in the fasted group. * P < 0.05 relative to control, # P < 0.05 relative to control; control n = 13 cells in 7 slices from 2 animals; ad libitum n = 6 cells in 3 slices from 1 animal; fasted n = 6 cells in 3 slices from 1 animal; one‐way ANOVA/LSD. Bars represent means and lines 1 SD of the change in membrane current obtained from a mixture of male and OVX female animals balanced across the different treatment and energy balance conditions. K–O , representative photomicrographs. K and L , low power (×4) image of eGFP‐fluorescing POMC ARC neurones and N/OFQ ARC mCherry fluorescing neurones ( K ) and fibres ( L ). M and N , high power (×40) DIC image of a recorded POMC ARC neurone ( M ) and eGFP fluorescence of the same neurone ( N ). O , high power (×40) image of mCherry‐fluorescing N/OFQ ARC cell bodies and fibres within the same vicinity of the recorded POMC ARC neurone. [Colour figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Cre recombinase‐dependent AAV vectors containing either cation channel rhodopsin‐2 (ChR2; Mattis et al., ; AAV1.EF1a.DIO.ChR2 [E123A].YFP.WPRE.jGH; 7.2 × 10 12 genomic copies/ml; 300 nl total volume; Addgene (Watertown, MA, USA) plasmid no. 35507), excitatory designer receptor exclusively activated by designer drug (DREADD; pAAV‐hSyn‐DIO‐hM3D(Gq)‐mCherry; 7×10 12 genomic copies/ml; 300 nl total volume; Addgene plasmid gifted by Bryan Roth, no. 44361), inhibitory DREADD (pAAV‐hSyn‐DIO‐hM4D(Gi)‐mCherry; 7 × 10 12 genomic copies/ml; 300 nl total volume; Addgene plasmid gifted by Bryan Roth, no. 44362), or their respective enhanced yellow fluorescent protein (eYFP; Mattis et al., ; pAAV‐Ef1a‐DIO EYFP; 1.0×10 13 ; 300 nl total volume; Addgene plasmid deposited by Karl Deisseroth, no. 27056) and mCherry blank controls (pAAV‐hSyn‐DIO‐mCherry; 7 × 10 12 genomic copies/ml; 300 nl total volume; Addgene plasmid gifted by Bryan Roth, no. 50459) were administered over a span of 2 min to double transgenic PNOC‐ cre /eGFP POMC mice, PNOC‐ cre mice and WT controls.

Techniques: Fluorescence, Inhibition, Control, Membrane